Dynamics and metal exchange properties of C4C4 RING domains from CNOT4 and the p44 subunit of TFIIH

Zinc fingers are small structured protein domains that require the coordination of zinc for a stable tertiary fold. Together with FYVE and PHD, the RING domain forms a distinct class of zinc-binding domains, where two zinc ions are ligated in a cross-braced manner, with the first and third pairs of ligands coordinating one zinc ion, while the second and fourth pairs ligate the other zinc ion. To investigate the relationship between the stability and dynamic behaviour of the domains and the stability of the metal-binding site, we studied metal exchange for the C4C4 RING domains of CNOT4 and the p44 subunit of TFIIH. We found that Zn(2+)-Cd(2+) exchange is different between the two metal-binding sites in the C4C4 RING domains of the two proteins. In order to understand the origins of these distinct exchange rates, we studied the backbone dynamics of both domains in the presence of zinc and of cadmium by NMR spectroscopy. The differential stability of the two metal-binding sites in the RING domains, as reflected by the different metal exchange rates, can be explained by a combination of accessibility and an electrostatic ion interaction model. A greater backbone flexibility for the p44 RING domain as compared to CNOT4 may be related to the distinct types of protein-protein interactions in which the two C4C4 RING domains are involved.     

The zinc finger domain of the archaeal minichromosome maintenance protein is required for helicase activity

The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication. The archaeon Methanobacterium thermoautotrophicum Delta H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme. The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX(2)CX(n)CX(2)C (C(4)) type. In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli. The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme. The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity. These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif.